591 research outputs found
The Duties of the City Veterinarian
The City Veterinarian position requires a variety of qualifications and skills. The basic requirements are a Doctor of Veterinary Medicine degree and two years of Public Health Inspection experience
Cryptotomography: reconstructing 3D Fourier intensities from randomly oriented single-shot diffraction patterns
We reconstructed the 3D Fourier intensity distribution of mono-disperse
prolate nano-particles using single-shot 2D coherent diffraction patterns
collected at DESY's FLASH facility when a bright, coherent, ultrafast X-ray
pulse intercepted individual particles of random, unmeasured orientations. This
first experimental demonstration of cryptotomography extended the
Expansion-Maximization-Compression (EMC) framework to accommodate unmeasured
fluctuations in photon fluence and loss of data due to saturation or background
scatter. This work is an important step towards realizing single-shot
diffraction imaging of single biomolecules.Comment: 4 pages, 4 figure
Different forms of African cassava mosaic virus capsid protein within plants and virions
One geminiviral gene encodes the capsid protein (CP), which can appear as several bands after electrophoresis depending on virus and plant. African cassava mosaic virus-Nigeria CP in Nicotiana benthamiana, however, yielded one band (~âŻ30âŻkDa) in total protein extracts and purified virions, although its expression in yeast yielded two bands (~âŻ30, 32âŻkDa). Mass spectrometry of the complete protein and its tryptic fragments from virions is consistent with a cleaved start M1, acetylated S2, and partial phosphorylation at T12, S25 and S62. Mutants for additional potentially modified sites (N223A; C235A) were fully infectious and formed geminiparticles. Separation in triton acetic acid urea gels confirmed charge changes of the CP between plants and yeast indicating differential phosphorylation. If the CP gene alone was expressed in plants, multiple bands were observed like in yeast. A high turnover rate indicates that post-translational modifications promote CP decay probably via the ubiquitin-triggered proteasomal pathway
Microinjection of antibodies targeting the lamin A/C histone-binding site blocks mitotic entry and reveals separate chromatin interactions with HP1, CenpB and PML
Lamins form a scaffold lining the nucleus that binds chromatin and contributes to spatial genome organization; however, due to the many other functions of lamins, studies knocking out or altering the lamin polymer cannot clearly distinguish between direct and indirect effects. To overcome this obstacle, we specifically targeted the mapped histone-binding site of A/C lamins by microinjecting antibodies specific to this region predicting that this would make the genome more mobile. No increase in chromatin mobility was observed; however, interestingly, injected cells failed to go through mitosis, while control antibody-injected cells did. This effect was not due to crosslinking of the lamin polymer, as Fab fragments also blocked mitosis. The lack of genome mobility suggested other lamin-chromatin interactions. To determine what these might be, mini-lamin A constructs were expressed with or without the histone-binding site that assembled into independent intranuclear structures. HP1, CenpB and PML proteins accumulated at these structures for both constructs, indicating that other sites supporting chromatin interactions exist on lamin A. Together, these results indicate that lamin A-chromatin interactions are highly redundant and more diverse than generally acknowledged and highlight the importance of trying to experimentally separate their individual functions
Influence of pump laser fluence on ultrafast structural changes in myoglobin
High-intensity femtosecond pulses from an X-ray free-electron laser enable pump probe experiments for investigating electronic and nuclear changes during light-induced reactions. On time scales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer. However, all ultra-fast TR-SFX studies to date have employed such high pump laser energies that several photons were nominally absorbed per chromophore. As multiphoton absorption may force the protein response into nonphysiological pathways, it is of great concern whether this experimental approach allows valid inferences to be drawn vis-a-vis biologically relevant single-photon-induced reactions. Here we describe ultrafast pump-probe SFX experiments on photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics) are seen to depend strongly on pump laser energy. Our results confirm both the feasibility and necessity of performing TR-SFX pump probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing design and interpretation of ultrafast TR-SFX pump probe experiments such that biologically relevant insight emerges
Electronic damage in S atoms in a native protein crystal induced by an intense X-ray free-electron laser pulse
Current hard X-ray free-electron laser (XFEL) sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed
Viscous hydrophilic injection matrices for serial crystallography
Serial (femtosecond) crystallography at synchrotron and X-ray free-electron
laser (XFEL) sources distributes the absorbed radiation dose over all crystals
used for data collection and therefore allows measurement of radiation damage
prone systems, including the use of microcrystals for room-temperature
measurements. Serial crystallography relies on fast and efficient exchange of
crystals upon X-ray exposure, which can be achieved using a variety of
methods, including various injection techniques. The latter vary significantly
in their flow rates â gas dynamic virtual nozzle based injectors provide very
thin fast-flowing jets, whereas high-viscosity extrusion injectors produce
much thicker streams with flow rates two to three orders of magnitude lower.
High-viscosity extrusion results in much lower sample consumption, as its
sample delivery speed is commensurate both with typical XFEL repetition rates
and with data acquisition rates at synchrotron sources. An obvious viscous
injection medium is lipidic cubic phase (LCP) as it is used for in meso
membrane protein crystallization. However, LCP has limited compatibility with
many crystallization conditions. While a few other viscous media have been
described in the literature, there is an ongoing need to identify additional
injection media for crystal embedding. Critical attributes are reliable
injection properties and a broad chemical compatibility to accommodate samples
as heterogeneous and sensitive as protein crystals. Here, the use of two novel
hydroÂgels as viscous injection matrices is described, namely sodium
carbÂoxyÂmethyl cellulose and the thermo-reversible block polymer Pluronic
F-127. Both are compatible with various crystallization conditions and yield
acceptable X-ray background. The stability and velocity of the extruded stream
were also analysed and the dependence of the stream velocity on the flow rate
was measured. In contrast with previously characterized injection media, both
new matrices afford very stable adjustable streams suitable for time-resolved
measurements
crystal and solution structures of the multidomain cochaperone DnaJ
Hsp70 chaperones assist in a large variety of protein-folding processes in the
cell. Crucial for these activities is the regulation of Hsp70 by Hsp40
cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP
hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but
is also known to possess chaperone activity of its own. The first structure of
a complete functional dimeric DnaJ was determined and the mobility of its
individual domains in solution was investigated. Crystal structures of the
complete molecular cochaperone DnaJ from Thermus thermophilus comprising the
J, GF and C-terminal domains and of the J and GF domains alone showed an
ordered GF domain interacting with the J domain. Structure-based EPR spin-
labelling studies as well as cross-linking results showed the existence of
multiple states of DnaJ in solution with different arrangements of the various
domains, which has implications for the function of DnaJ.1\. Auflag
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